NLRP2 is a suppressor of NF-ƙB signaling and HLA-C expression in human trophoblasts†,‡

Abstract

During pregnancy, fetal extravillous trophoblasts (EVT) play a key role in the regulation of maternal T cell and NK cell responses. EVT display a unique combination of human leukocyte antigens (HLA); EVT do not express HLA-A and HLA-B, but do express HLA-C, HLA-E, and HLA-G. The mechanisms establishing this unique HLA expression pattern have not been fully elucidated. The major histocompatibility complex (MHC) class I and class II transcriptional activators NLRC5 and CIITA are expressed neither by EVT nor by the EVT model cell line JEG3, which has an MHC expression pattern identical to that of EVT. Therefore, other MHC regulators must be present to control HLA-C, HLA-E, and HLA-G expression in these cells. CIITA and NLRC5 are both members of the nucleotide-binding domain, leucine-rich repeat (NLR) family of proteins. Another member of this family, NLRP2, is highly expressed by EVT and JEG3, but not in maternal decidual stromal cells. In this study, transcription activator-like effector nuclease technology was used to delete NLRP2 in JEG3. Furthermore, lentiviral delivery of shRNA was used to knockdown NLRP2 in JEG3 and primary EVT. Upon NLRP2 deletion, Tumor Necrosis Factor-α (TNFα)-induced phosphorylation of NF-KB p65 increased in JEG3 and EVT, and more surprisingly a significant increase in constitutive HLA-C expression was observed in JEG3. These data suggest a broader role for NLR family members in the regulation of MHC expression during inflammation, thus forming a bridge between innate and adaptive immune responses. As suppressor of proinflammatory responses, NLRP2 may contribute to preventing unwanted antifetal responses.

Keywords: HLA-G; MHC; NOD-like receptor; inflammasome; pregnancy.

Figure 1.

High expression of NLRP2, but not NLRC5 in EVT and JEG3. (A) Heat map depicts the global mRNA expression levels for all members of the NLR family in VT, EVT, JEG3, and DSC as found in (2). (B) Table shows MHC protein expression profile and the presence of MHC transcriptional regulators in the four cell types used. Graphs depict mRNA expression levels of (C) NLRC5, (D) NLRP12, and (E) NLRP2 in VT, EVT, JEG3, and DSC. Bars indicate median expression values.

Figure 2.

NLRP2 in JEG3 and EVT is not localized in the nucleus. (A) Confocal images of JEG3. Panels depict NLRP2 (top) and IgG2a (bottom), DAPI, F-actin, and composite images. (B) Confocal images of two isolates of primary EVT (EVT1 and EVT2). Panels depict NLRP2, HLA-G, DAPI, and composite image for EVT1 and NLRP2, DAPI and composite image for EVT2.

Figure 3.

HLA-C protein and mRNA expression is increased in NLRP2 knockout JEG3 clones. Graphs depict mean fluorescence intensity (MFI) for (A) HLA-C (mAb TRA2G9), (B) HLA-C (mAb DT9), (C) HLA-E (mAb 3D12), and (D) HLA-G (mAb MEM-G9) protein levels on parental JEG3 (JEG3) as well as on wild-type (WT) heterozygote (HZ), knockout (KO) clones. Five technical replicates for JEG3, four WT clones, and four HZ clones with a minimum of two replicates each, two replicates for four HZ clones and six replicates for KO clone 1 are depicted. (E) Representative FACS plots of HLA-C, HLA-E, and HLA-G on the parental and targeted cell lines. The lines indicate mean expression of HLA-C, HLA-E, and HLA-G in untreated JEG3 to visualize the shift in HLA-C but not HLA-E and HLA-G in HZ and KO cells. Relative mRNA expression of (F) HLA-C, (G) HLA-E, and (H) HLA-G normalized to GAPDH on wild-type (WT) heterozygote (HZ) and knockout (KO) JEG3 clones. Bars indicate median values and SEM. **P < 0.01.

Figure 4.

Major histocompatibility complex class I expression on NLRP2 clones upon IFNγ or TNFα stimulation. (A) Graph depicts mean fluorescence intensity (MFI) of HLA-C (mAb clone DT9) protein expression on JEG3, wild-type (WT) heterozygote (HZ), and knockout (KO) clones in the absence of stimulation or stimulated with IFNγ (100 ng/ml) or TNFα (20 ng/ml). Relative protein expression for (B) HLA-C, (C) HLA-E, (D) HLA-G, and (E) W6/32. Relative protein expression levels are plotted as expression on stimulated cells relative to unstimulated cells. Graphs depict median and interquartile range of a least five independent experiments. *P < 0.05 **P < 0.01.

Figure 5.

Deletion of NLRP2 increases NF-ƙB p65-P upon TNFα stimulation. (A) Western blot images of NF-κB p65-P and HSP70 protein expression in NLRP2 wild-type (WT) and knockout (KO) JEG3 clones stimulated with 20 ng/ml TNFα for 0, 5, 15, 30, and 45 min. (B) Western blot images of HSP70, NF-κB p65, and IκBα expression in NLRP2 wild-type (WT, left) and knockout (KO, right) JEG3 clones stimulated with 20 ng/ml TNFα for 0, 5, 15, and 30 min. (C) Western blot quantification of NF-κB p65-P, NF-κB p65, and IκBα relative to HSP70 expression in NLRP2 wild-type (gray bars) and knockout (black bars) JEG3 clones stimulated with 20 ng/ml TNFα for 0, 5, 15, 30, and 45 min of one representative sample. (D) Graphs depict the fold change (FC) of NF-κB p65-P, NF-κB p65, and IκBα protein in KO clones relative to WT clones.

Figure 6.

NLRP2 knockdown in primary EVT increases phosphorylation of NF-κB p65. (A) Western blots of NF-κB p65-P, NLRP2, HSP70, and HLA-G protein expression in primary EVT transduced with scrambled (SC) and NLRP2 (KD) shRNA stimulated without or with 20 ng/ml TNFα for 15 min. Graphs depict (B) NF-κB p65-P, (C) NLRP2, and (D) HLA-G relative to HSP70 in two primary EVT (EVT1 and EVT2) isolates transduced with scrambled (SC) and NLRP2 (KD) shRNA. NF-κB p65-P is depicted without or with 20 ng/ml TNFα for 15 min.